1. Field of the Invention
The present invention relates to a method for amplifying a nucleic acid (DNA or RNA), and more particularly, to an isothermal nucleic acid amplification method performed in combination with the LAMP method using a heat resistant ligase and DNA polymerase having strand displacement activity.
2. Background Art
In the life phenomenon research, amplification of DNA and RNA has been used for various purposes. As examples of a method for gene expression analysis and a method for qualification of gene expression level, a competitive PCR method (A. Wang, et al., Proc Natl Acad Sci USA, 86, 9717-9721(1989) and a real-time PCR method (K. Edwards, et al., Journal of Antimicrobial, 54, 968 (2004) etc. are known. They each employ a general nucleic acid amplification method, PCR (polymerase chain reaction) method (R. K. Saiki, et al., Science, 239, 487-491 (1988)) to determine the expression level of a gene based on the amplified gene.
The nucleic acid amplification method for use in the aforementioned analyses is constituted of three steps: denaturing double stranded DNA into a single stranded DNA, annealing to hybridize a primer with the single stranded DNA, elongating the primer to form a complementary chain, or constituted of two steps: denaturation and elongation. In both cases, a cycle consisting of a high temperature step and a low temperature step is inevitably performed repeatedly. This cycle of the PCR method must be performed by use of a thermal cycler capable of accurately controlling temperature. Furthermore, the time required for adjusting the temperature of an apparatus and a reaction solution to a predetermined value increases as the number of cycles increases. Consequently, it takes long time for analysis.
Then, to overcome the aforementioned problems, a method has been developed for amplifying a nucleic acid at isothermal conditions. As examples of such an isothermal nucleic acid amplification method, the following are principally known:
NASBA (Nucleic Acid Sequence-Based Amplification) method (J. Compton, et. al., Nature, 350, 91-92 (1991);
SDA (strand displacement amplification) method (G. T. Walker, et. al., Proc. Natl. Acad. Sci USA, 89, 392-396 (1992));
3SR (self-sustained sequence replication) method (J. C. Guatelli, et al., Proc Natl Acad Sci USA, 87, 1874-1878 (1990));
TMA (transcription-mediated amplification) method (JP Patent No. 3241717);
QB replicase amplification method (JP Patent No. 2710159); and
LAMP (loop-mediated isothermal amplification) method (JP Patent No. 3313358 and T. Notomi, et al., Nucleic Acids Research, 28, e63 (2000)).
In these isothermal nucleic acid amplification methods, elongation of a primer, annealing of the primer to hybridize with a single stranded elongation product, and the following elongation of the primer are performed in a reaction mixture maintained at a constant temperature.
Of these isothermal nucleic acid amplification methods, an SDA method and a LAMP method employ DNA polymerase having strand displacement activity. In the SDA method, dATPαS is used as a substrate in place of dATP to provide a priming site serving as an amplification initiation point. In the LAMP method, a primer elongation product is designed to form a self-looped structure. The SDA method has a problem in that an efficiency of an enzyme for incorporating a substrate is low, decreasing amplification efficiency. The LAMP method has a problem in that it is difficult to design a primer. More specifically, it is difficult to design optimal primers in accordance with various detection items. In the circumstances, it has been desired to develop an isothermal nucleic acid amplification method to overcome these problems.